Rote of adenosine triphosphatase, phospholipids, and vesicular structure in the calcification of isolated and reconstituted matrix vesicles

The electron-microscopic cytochemical localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) was determined in chick epiphyseal growth-plate cartilage. In the reserve zone, mitochondria and lysosomes contained substantial amounts of reaction product, while the plasma membrane and the Golgi complex showed very weak enzymatic activity, and matrix vesicle membranes did not exhibit the cytochemical reaction. As maturation proceeded, the plasma membrane, Golgi complex, and matrix vesicle membranes also stained and were most intense in the proliferative and early hypertrophic zones. From the hypertrophic to the calcifying zone, cytochemical staining decreased progressively in the plasma membrane, the Golgi complex, and lysosomes, while in some cases mitochondrial reaction product remained intense. Matrix vesicles lost their enzymatic activity at the same time that matrix vesicle calcification commenced. It is proposed that this event allows matrix vesicles to calcify, since efflux of calcium would no longer occur.

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Electron microscopic localization of adenosine triphosphate (ATP)-hydrolyzing activity in isolated matrix vesicles and reconstituted vesicles from calf cartilage.

Journal of Histochemistry & Cytochemistry ◽

10.1177/31.4.6219157 ◽

1983 ◽

Vol 31 (4) ◽

pp. 462-470 ◽

Cited By ~ 16

Author(s):

S Kanabe ◽

H H Hsu ◽

R N Cecil ◽

H C Anderson

Keyword(s):

Atpase Activity ◽

Phosphatase Activity ◽

Adenosine Triphosphatase ◽

Matrix Vesicles ◽

Matrix Vesicle ◽

Epiphyseal Growth Plate ◽

Vesicle Membrane ◽

Electron Microscopic ◽

The Matrix ◽

Electron Microscopic Localization

The presence and distribution of adenosine triphosphatase (ATPase) activity in isolated matrix vesicles and reconstituted vesicles from fetal calf epiphyseal growth plate cartilage was studied by electron microscopic cytochemical methods to determine whether phosphatase activity would be found concentrated on the inside or the outside of matrix vesicle membranes or on both sides, and whether reconstitution of vesicles from deoxycholate-solubilized substituents would lead to the reassembly of membranes with ATPase incorporated. ATPase activity was observed on both the outer and inner surfaces of the investing membranes of isolated matrix vesicles and reconstituted vesicles. A transmembrane location of ATPase could indicate phosphate transfer across the vesicle membrane. Orthophosphate released by phosphatase activity within the protected microenvironment of the matrix vesicle could combine with membrane- or lipid-bound calcium, known to be present in vesicles, to form the first hydroxyapatite mineral during calcification.

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Freeze-etch studies of epiphyseal growth plate cartilage reveal matrix vesicles associated with calcification

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084223 ◽

1978 ◽

Vol 36 (2) ◽

pp. 670-671

Author(s):

Russell N. A. Cecil ◽

H. Clarke Anderson

Keyword(s):

Chromic Acid ◽

Light And Electron Microscopy ◽

Matrix Vesicles ◽

Matrix Vesicle ◽

Glycerol Solution ◽

Sprague Dawley ◽

Epiphyseal Growth Plate ◽

Growth Plates ◽

Sprague Dawley Rats ◽

Tibial Epiphyseal

Unfixed proximal tibial epiphyseal growth plates were studied by freeze-etch to confirm the presence of extracellular calcifying matrix vesicles and to determine the substructure of matrix vesicle membranes as compared to plasma and other membranes of intact chondrocytes. Growth plates from 6-10 week old Sprague-Dawley rats were cut into 1x3 mm blocks whose long dimension was oriented either perpendicular or parallel to the long axis of the tibia. Some blocks were fixed at pH 7. 0 in 0. 2M cacodylate - buffered 2. 5% glutaraldehyde for 1 hour at 4ÅC. The blocks were immersed in 30% glycerol solution at 4ÅC for 1 hour, frozen in liquid nitrogen, and then fractured, etched for 2 minutes, and coated with platinum, carbon and 0. 2% Formvar solution. The replicas were cleaned with chromic acid, floated onto Formvar coated grids, and examined with a Phillips EM 300 electron microscope.Fixed and unfixed specimens appeared similar in ultrastructure. Chondrocytes, matrix, and matrix vesicles were identified. In specimens fractured parallel to the long axis of the tibia, the reserve, proliferative, hypertrophic, and calcifying zones could be discerned as described by light and electron microscopy.

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Effects of EDTA, Hyaluronidase and Collagenase on Epiphyseal Cartilage Matrix

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100020 ◽

1968 ◽

Vol 26 ◽

pp. 56-57

Author(s):

H. Clarke Anderson ◽

Priscilla R. Coulter

Keyword(s):

Light And Electron Microscopy ◽

Matrix Vesicles ◽

Cartilage Matrix ◽

Collagen Fibrils ◽

Epiphyseal Cartilage ◽

Dense Matrix ◽

Type Iv ◽

Bovine Testicular Hyaluronidase ◽

The Matrix ◽

Testicular Hyaluronidase

Epiphyseal cartilage matrix contains fibrils and particles of at least 5 different types: 1. Banded collagen fibrils, present throughout the matrix, but not seen in the lacunae. 2. Non-periodic fine fibrils <100Å in diameter (Fig. 1), which are most notable in the lacunae, and may represent immature collagen. 3. Electron dense matrix granules (Fig. 1) which are often attached to fine fibrils and collagen fibrils, and probably contain protein-polysaccharide although the possibility of a mineral content has not been excluded. 4. Matrix vesicles (Fig. 2) which show a selective distribution throughout the epiphysis, and may play a role in calcification. 5. Needle-like apatite crystals (Fig. 2).Blocks of formalin-fixed epiphysis from weanling mice were digested with the following agents in 0.1M phosphate buffer: a) 5% ethylenediaminetetraacetate (EDTA) at pH 8.3, b) 0.015% bovine testicular hyaluronidase (Sigma, type IV, 750 units/mg) at pH 5.5, and c) 0.1% collagenase (Worthington, chromatograhically pure, 200 units/mg) at pH 7.4. All digestions were carried out at 37°C overnight. Following digestion tissues were examined by light and electron microscopy to determine changes in the various fibrils and particles of the matrix.

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Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094401 ◽

1972 ◽

Vol 30 ◽

pp. 230-231

Author(s):

James Cronshaw ◽

Jamison E. Gilder

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Adenosine Triphosphatase ◽

Higher Plants ◽

High Surface ◽

Root Tip ◽

Animal Cells ◽

Physiological Processes ◽

Tip Cells ◽

Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

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Effects of Hypophysectomy on the Occurrence and Calcifiability of Matrix Vesicles in the Epiphyseal Growth Plate of Immature Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002579 ◽

1980 ◽

Vol 38 ◽

pp. 578-579

Author(s):

S. I. Coleman ◽

W. J. Dougherty

Keyword(s):

Growth Plate ◽

Matrix Vesicles ◽

Epiphyseal Growth Plate ◽

Hard Tissue ◽

Hormonal Factors ◽

Post Surgery ◽

Mineral Deposition ◽

Thin Sectioning ◽

Integral Role ◽

Cellular Secretion

In the cellular secretion theory of mineral deposition, extracellular matrix vesicles are believed to play an integral role in hard tissue mineralization (1). Membrane limited matrix vesicles arise from the plasma membrane of epiphyseal chondrocytes and tooth odontoblasts by a budding process (2, 3). Nutritional and hormonal factors have been postulated to play essential roles in mineral deposition and apparently have a direct effect on matrix vesicles of calcifying cartilage as concluded by Anderson and Sajdera (4). Immature (75-85 gm) Long-Evans hooded rats were hypophysectomized by the parapharyngeal approach and maintained fourteen (14) days post-surgery. At this time, the animals were anesthetized and perfusion fixed in cacodylate buffered 2.5% glutaraldehyde. The proximal tibias were quickly dissected out and split sagittally. One half was used for light microscopy (LM) and the other for electron microscopy (EM). The halves used for EM were cut into blocks approximately 1×3 mm. The tissue blocks were prepared for ultra-thin sectioning and transmission EM. The tissue was oriented so as to section through the epiphyseal growth plate from the zone of proliferating cartilage on down through the hypertrophic zone and into the initial trabecular bone. Sections were studied stained (double heavy metal) and unstained.

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